L32 Gene Knockout And Overexpression Strains And Growth Characteristics

The evolutionarily conservative ribosomal protein L32 exists widely in different biological groups such as animals, plants and yeast and so on. Most of the eukaryotic ribosomal protein L32 is only encoded by one gene, but the Schizosaccharomyces pombe ribosomal protein L32 is encoded by two genes which respectively express ribosomal protein L32-1 and L32-2. In our laboratory,we have not only cloned and expressed 132-2 gene,but also done researches in its functions before to demonstrate that maybe it is a transcriptional regulation factor. Considering their high amino acid similarity of L32-1 and L32-2,that is 96.85%,we try to discover and conform their functions. Therefore, this thesis does researches in gene cloning, gene knockout,and growth features of overexpression or knockout of 132 gene.In this thesis, wild Schizosaccharomyces pombe genome DNA was extracted, and then,132-1 gene was synthesized with specific primers by PCR.132-1 was ligated into pMD18-T vector,creating plasmid pMD18-T/ι32-1. After that,ι32-1 gene was digested from pMD18-T/ι32-1 by restriction endonuclease enzyme,then, ligated into pESP-3 vector to creat plasmid pESP-3/ι32-1. The recombinant plasmid was transformed into SP-Q01 cell to construct expression strain SP-Q01 (pESP-3/ι32-1).Besides, expression strain SP-Q01 (pESP-3/ι32-2) has already been constructed in our labs before.The 132 gene knockout strains SP-Q01(Δι32-1) and SP-Q01(Δι32-2) were also costructed in this thesis. Through designing three pairs specific primers and two turns over-lap PCR reactions,we successfully required fusion gene cosist of the upstream and downstream gene of 132.After that,we transformed the gene into SP-Q01 cell, by the theory of homology exchange, G418 screening, we successfully constructed yeast strains of SP-Q01(Δι32-1) and SP-Q01(Δι32-2).Besides,based on the konckout strains, we successfully complemented related genes into them to constrct SP-Q01 (Δι32-1, pESP-3/ι32-1), SP-Q01 (Δι32-1,pESP-3/ι32-2), SP-Q01 (Δι32-2,pESP-3/ι32-1), SP-Q01 (Δι32-2,pESP-3/ι32-2) yeast strains.Finally, growth features of over-expression or knockout of 132 gene were studied.The differences of the growth curve showed thatι32-1 overexpression strain SP-Q01 (pESP-3/ι32-1) grows the slowest; Through the observation of their modalities, phenomenon of flocculation appeared in the 132 gene knockout strains SP-Q01 (Δι32-1) and SP-Q01 (Δι32-2); Besides,large cells appeared in the overexpression strains SP-Q01 (pESP-3/ι32-1) and SP-Q01 (pESP-3/ι32-2).Intensive studies were needed to explain all of the above.